The long-term objective of this research is defining the mechanism by which GTP induces assembly of tubulin into microtubules. This task requires complete characterization or mapping of the nucleotide binding site of tubulin. To achieve this goal, the identity of functional groups of tubulin that comprise the nucleotide site, their geometric arrangement, the nuclei of GTP that interect with the functional groups and the role of Mg(II) in binding and assembly must be established. This research addresses the role of Mg(II) and the identity of GTP moieties essential to binding and assembly through three specific aims. First, dissociation constants of GTP and the inhibitor GDP will be measured on tubulin from which all exchangeable nucleotide has been removed. Second, the necessity of Mg(II) for nucleotide binding and tubulin assembly is to be investigated with GTP, GDP and Cr(III)GTP. The geometric and stereochemical properties of the nucleotide site will be studied using chromiun and phosphorothiote diastereomeric analogs of GTP and GDP. Nucleotide removal will be accomplished by treatment with charcoal or alkaline phosphatase. Dissociation constants will be measured by centrifugal ultrafiltration and centrifugal gel filtration, using radioactively labeled nucleotides. Assembly of tubulin will be measured by the increase in absorbance due to scattered light at 350nm and verified by electron microscopy. The results of this study could have health ramifications at the molecular level since microtubules are implicated in so many cellular functions, such as maintenance of cell shape, intracellular movement, and secretory function. Malfunctioning of microtubules could lead to disease; and abnormality in properties, in assembly, and in amounts of cellular tubulin have already been linked to some pathological conditions (cystic fibrosis, diabetes, and phenylketonuria).